Plant transcription factors

ABSTRACT

This invention relates to an isolated nucleic acid fragment encoding a transcription factor. The invention also relates to the construction of a chimeric gene encoding all or a portion of the transcription factor, in sense or antisense orientation, wherein expression of the chimeric gene results in production of altered levels of accumulated oil in a transformed host cell.

This application is a continuation of U.S. patent application Ser. No.11/899,370, filed Sep. 4, 2007, now U.S. Pat. No. 7,888,560, issued Feb.15, 2011, which is a continuation of the national stage entry ofPCT/US01/50908, filed Oct. 24, 2001, now abandoned, which claims thebenefit of U.S. Provisional Application No. 60/242,739, filed Oct. 24,2000, each of which are hereby incorporated by reference.

FIELD OF THE INVENTION

This invention is in the field of plant molecular biology. Morespecifically, this invention pertains to nucleic acid fragments encodingtranscription factors in plants and seeds.

BACKGROUND. OF THE INVENTION

Higher plant embryogenesis is divided conceptually into two distinctphases: early morphogenic processes that give rise to embryonic celltypes, tissues, and organ systems, and late maturation events that allowthe fully developed embryo to enter a desiccated and metabolicallyinactive state. Upon reception of the appropriate signals, the dormantembryo germinates, and seedling development begins. Thus, seedmaturation and metabolic quiescence interrupt the morphogeneticprocesses that occur during embryogenesis and seedling development. Thisunique form of development underlies, in part, a plant's ability to makeseeds, a trait that has conferred significant selective advantages tohigher plants. Because lower plants do not make seeds and do not undergoembryo maturation, this bipartite mode of embryogenesis is thought tohave resulted from the insertion of mutation events into the higherplant life cycle. Little is known at the mechanistic level about howdistinct processes that occur during the morphogenesis and seedmaturation phases are coordinated.

The leafy cotyledon) (LEC1) gene controls many distinct aspects ofembryogenesis. The lec1 mutation is pleiotropic, which suggest that LEC1has several roles in late embryo development. For example, LEC1 isrequired for specific aspects of seed maturation, inhibiting prematuregermination and plays a role in the specification of embryonic organidentity. Finally, LEC1 appears to act only during embryo development.Two other LEC class genes, LEC2 and FUSCA3 (FUS3), are thought to sharesimilar or overlapping functions with LEC1, including the specificationof cotyledon identity and the maintenance of maturation. It is unknownhow LEC class genes act at the molecular level, but their involvement inmany diverse aspects of embryogenesis suggests that these genes encodeproducts that serve as regulators of higher plant embryonic processes.The LEC1-related transcription factors disclosed below all have homologyto the maize CAAT-box DNA binding protein subunit B and the ArabidopsisLEC1 protein (WO 9837184-A) and as such may define a new family of LEC1transcription factors.

Accordingly, the availability of nucleic acid sequences encoding all ora portion of these LEC1-related transcription factors would facilitatestudies to better understand plant embryogenesis, and provide genetictools for the manipulation of plant growth.

SUMMARY OF THE INVENTION

In a first embodiment, the present invention concerns an isolatedpolynucleotide comprising: (a) a nucleotide sequence encoding apolypeptide comprising at least 50 or 100 amino acids, wherein the aminoacid sequence of the polypeptide and the amino acid sequence of OF SEQID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30 have atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity basedon the Clustal alignment method, or (b) the complement of the nucleotidesequence, wherein the complement and the nucleotide sequence contain thesame number of nucleotides and are 100% complementary. The polypeptidepreferably comprises the amino acid sequence of OF SEQ ID NO:2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30. The nucleotide sequencepreferably comprises the nucleotide sequence of SEQ ID NO:1, 3, 5, 7, 9,11, 13, 15, 17, 19, 21, 23, 25, 27, or 29. The polypeptide preferably isa Lec1-related transcription factor.

In a second embodiment, the present invention relates to a chimeric genecomprising any of the isolated polynucleotides of the present inventionoperably linked to a regulatory sequence, and a cell, a plant, and aseed comprising the chimeric gene.

In a third embodiment, the present invention relates to a vectorcomprising any of the isolated polynucleotides of the present invention.

In a fourth embodiment, the present invention relates to an isolatedpolynucleotide comprising a nucleotide sequence comprised by any of thepolynucleotides of the first embodiment, wherein the nucleotide sequencecontains at least 30, 40, or 60 nucleotides, and a cell, a plant, and aseed comprising the isolated polynucleotide.

In a fifth embodiment, the present invention relates to a method fortransforming a cell comprising transforming a cell with any of theisolated polynucleotides of the present invention, and the celltransformed by this method. Advantageously, the cell is eukaryotic,e.g., a yeast or plant cell, or prokaryotic, e.g., a bacterium.

In a sixth embodiment, the present invention relates to a method forproducing a transgenic plant comprising transforming a plant cell withany of the isolated polynucleotides of the present invention andregenerating a plant from the transformed plant cell, the transgenicplant produced by this method, and the seed obtained from thistransgenic plant.

In a seventh embodiment, the present invention concerns an isolatedpolypeptide comprising an amino acid sequence comprising at least 50 or100 amino acids, wherein the amino acid sequence and the amino acidsequence of OF SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28 or 30 have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity based on the Clustal alignment method. The amino acidsequence preferably comprises the amino acid sequence of OF SEQ ID NO:2,4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30. The polypeptidepreferably is a Lec1-related transcription factor.

In an eight embodiment, this invention relates to a method for isolatinga polypeptide encoded by any of the polynucleotides of the firstembodiment comprising transforming a cell with the polynucleotide,causing the polypeptide to be produced in the transformed cell, andisolating the polypeptide from the transformed cell.

In a ninth embodiment, the present invention relates to a virus,preferably a baculovirus, comprising any of the isolated polynucleotidesof the present invention or any of the chimeric genes of the presentinvention.

In a tenth embodiment, the invention relates to a method of selecting anisolated polynucleotide that affects the level of expression of a geneencoding a Lec1-related transcription factor protein or activity in ahost cell, preferably a plant cell, the method comprising the steps of:(a) constructing an isolated polynucleotide of the present invention oran isolated chimeric gene of the present invention; (b) introducing theisolated polynucleotide or the isolated chimeric gene into a host cell;(c) measuring the level of the Lec1-related transcription factor proteinor activity in the host cell containing the isolated polynucleotide; and(d) comparing the level of the Lec1-related transcription factor proteinor activity in the host cell containing the isolated polynucleotide withthe level of the Lec1-related transcription factor protein or activityin the host cell that does not contain the isolated polynucleotide.

In an eleventh embodiment, the invention concerns a method of obtaininga nucleic acid fragment encoding a substantial portion of a Lec1-relatedtranscription factor protein, preferably a plant Lec1-relatedtranscription factor protein comprising the steps of: synthesizing anoligonucleotide primer comprising a nucleotide sequence of at least oneof 30 (preferably at least one of 40, most preferably at least one of60) contiguous nucleotides derived from a nucleotide sequence selectedfrom the group consisting of SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19,21, 23, 25, 27, and 29, and the complement of such nucleotide sequences;and amplifying a nucleic acid fragment (preferably a cDNA inserted in acloning vector) using the oligonucleotide primer. The amplified nucleicacid fragment preferably will encode a substantial portion of aLec1-related transcription factor protein amino acid sequence.

In a twelfth embodiment, this invention relates to a method of obtaininga nucleic acid fragment encoding all or a substantial portion of theamino acid sequence encoding a Lec1-related transcription factor proteincomprising the steps of: probing a cDNA or genomic library with anisolated polynucleotide of the present invention; identifying a DNAclone that hybridizes with an isolated polynucleotide of the presentinvention; isolating the identified DNA clone; and sequencing the cDNAor genomic fragment that comprises the isolated DNA clone.

In a thirteenth embodiment, this invention concerns a method forpositive selection of a transformed cell comprising: (a) transforming ahost cell with the chimeric gene of the present invention or anexpression cassette of the present invention; and (b) growing thetransformed host cell, preferably a plant cell, such as a monocot or adicot, under conditions which allow expression of the Lec1-relatedtranscription factor polynucleotide in an amount sufficient tocomplement a null mutant to provide a positive selection means.

In a fourteenth embodiment, this invention relates to a method ofaltering the level of expression of a Lec1-related transcription factorprotein in a host cell comprising: (a) transforming a host cell with achimeric gene of the present invention; and (b) growing the transformedhost cell under conditions that are suitable for expression of thechimeric gene wherein expression of the chimeric gene results inproduction of altered levels of the Lec1-related transcription factorprotein in the transformed host cell.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTINGS

The invention can be more fully understood from the following detaileddescription and the accompanying drawings and Sequence Listing that forma part of this application.

FIG. 1 shows a comparison of the amino acid sequences of theLec1-related transcription factors from Momordica (SEQ ID NO:2, labeledSID2), eucalyptus (SID4), corn (SID 6 and 8), rice (SID 10 and 12),soybean (SID 14, 16, 18, 20, and 22), wheat (SID 24, 26, and 28), andCanna (SID30); compared to the closest prior art sequences fromArabidopsis (SID 31, 33, 34, and 35) and corn (SID32). Sequence elementsEQDRXLPIAN (SEQ ID NO:36) and QECVSEFISFXTXE (SEQ ID NO:37), where “X”denotes any amino acid, are contained within the active site region ofthe polypeptide and appear to be characteristic of Lec1-relatedtranscription factors.

Table 1 lists the polypeptides that are described herein, thedesignation of the cDNA clones that comprise the nucleic acid fragmentsencoding polypeptides representing all or a substantial portion of thesepolypeptides, and the corresponding identifier (SEQ ID NO:) as used inthe attached Sequence Listing. The sequence descriptions and SequenceListing attached hereto comply with the rules governing nucleotideand/or amino acid sequence disclosures in patent applications as setforth in 37 C.F.R. §1.821-1.825.

TABLE 1 LEC1-Related Transcription Factors SEQ ID NO: Protein CloneDesignation (Nucleotide) (Amino Acid) LEC-1-related fds.pk0003.h5 1 2transcription factor LEC-1-related eef1c.pk004.c8 3 4 transcriptionfactor LEC-1-related cbn10.pk0005.e6 5 6 transcription factorLEC-1-related p0006.cbysa51r 7 8 transcription factor LEC-1-relatedrl0n.pk0061.c8 9 10 transcription factor LEC-1-related rsl1n.pk002.g1011 12 transcription factor LEC-1-related ses4d.pk0037.e3 13 14transcription factor LEC-1-related src2c.pk003.i13 15 16 transcriptionfactor LEC-1-related src2c.pk011.m12 17 18 transcription factorLEC-1-related src2c.pk025.b3 19 20 transcription factor LEC-1-relatedsrc3c.pk028j21 21 22 transcription factor LEC-1-related wkm1c.pk0002.d723 24 transcription factor LEC-1-related wlk8.pk0001.e10 25 26transcription factor LEC-1-related wlm96.pk037.k9 27 28 transcriptionfactor LEC-1-related ect1c.pk007.p18:fis 29 30 transcription factor

The Sequence Listing contains the one letter code for nucleotidesequence characters and the three letter codes for amino acids asdefined in conformity with the IUPAC-IUBMB standards described inNucleic Acids Res. 13:3021-3030 (1985) and in the Biochemical J. 219(No. 2):345-373 (1984) which are herein incorporated by reference. Thesymbols and format used for nucleotide and amino acid sequence datacomply with the rules set forth in 37 C.F.R. §1.822.

DETAILED DESCRIPTION OF THE INVENTION

In the context of this disclosure, a number of terms shall be utilized.The terms “polynucleotide”, “polynucleotide sequence”, “nucleic acidsequence”, and “nucleic acid fragment”/“isolated nucleic acid fragment”are used interchangeably herein. These terms encompass nucleotidesequences and the like. A polynucleotide may be a polymer of RNA or DNAthat is single- or double-stranded, that optionally contains synthetic,non-natural or altered nucleotide bases. A polynucleotide in the form ofa polymer of DNA may be comprised of one or more segments of cDNA,genomic DNA, synthetic DNA, or mixtures thereof. An isolatedpolynucleotide of the present invention may include at least 30contiguous nucleotides, preferably at least 40 contiguous nucleotides,most preferably at least 60 contiguous nucleotides derived from SEQ IDNO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, or 29, or thecomplement of such sequences.

The term “isolated” refers to materials, such as nucleic acid moleculesand/or proteins, which are substantially free or otherwise removed fromcomponents that normally accompany or interact with the materials in anaturally occurring environment. Isolated polynucleotides may bepurified from a host cell in which they naturally occur. Conventionalnucleic acid purification methods known to skilled artisans may be usedto obtain isolated polynucleotides. The term also embraces recombinantpolynucleotides and chemically synthesized polynucleotides.

The term “recombinant” means, for example, that a nucleic acid sequenceis made by an artificial combination of two otherwise separated segmentsof sequence, e.g., by chemical synthesis or by the manipulation ofisolated nucleic acids by genetic engineering techniques.

As used herein, “contig” refers to a nucleotide sequence that isassembled from two or more constituent nucleotide sequences that sharecommon or overlapping regions of sequence homology. For example, thenucleotide sequences of two or more nucleic acid fragments can becompared and aligned in order to identify common or overlappingsequences. Where common or overlapping sequences exist between two ormore nucleic acid fragments, the sequences (and thus their correspondingnucleic acid fragments) can be assembled into a single contiguousnucleotide sequence.

As used herein, “substantially similar” refers to nucleic acid fragmentswherein changes in one or more nucleotide bases results in substitutionof one or more amino acids, but do not affect the functional propertiesof the polypeptide encoded by the nucleotide sequence. “Substantiallysimilar” also refers to nucleic acid fragments wherein changes in one ormore nucleotide bases does not affect the ability of the nucleic acidfragment to mediate alteration of gene expression by gene silencingthrough for example antisense or co-suppression technology.“Substantially similar” also refers to modifications of the nucleic acidfragments of the instant invention such as deletion or insertion of oneor more nucleotides that do not substantially affect the functionalproperties of the resulting transcript vis-à-vis the ability to mediategene silencing or alteration of the functional properties of theresulting protein molecule. It is therefore understood that theinvention encompasses more than the specific exemplary nucleotide oramino acid sequences and includes functional equivalents thereof. Theterms “substantially similar” and “corresponding substantially” are usedinterchangeably herein.

Substantially similar nucleic acid fragments may be selected byscreening nucleic acid fragments representing subfragments ormodifications of the nucleic acid fragments of the instant invention,wherein one or more nucleotides are substituted, deleted and/orinserted, for their ability to affect the level of the polypeptideencoded by the unmodified nucleic acid fragment in a plant or plantcell. For example, a substantially similar nucleic acid fragmentrepresenting at least 30 contiguous nucleotides, preferably at least 40contiguous nucleotides, most preferably at least 60 contiguousnucleotides derived from the instant nucleic acid fragment can beconstructed and introduced into a plant or plant cell. The level of thepolypeptide encoded by the unmodified nucleic acid fragment present in aplant or plant cell exposed to the substantially similar nucleicfragment can then be compared to the level of the polypeptide in a plantor plant cell that is not exposed to the substantially similar nucleicacid fragment.

For example, it is well known in the art that antisense suppression andco-suppression of gene expression may be accomplished using nucleic acidfragments representing less than the entire coding region of a gene, andby using nucleic acid fragments that do not share 100% sequence identitywith the gene to be suppressed. Moreover, alterations in a nucleic acidfragment which result in the production of a chemically equivalent aminoacid at a given site, but do not effect the functional properties of theencoded polypeptide, are well known in the art. Thus, a codon for theamino acid alanine, a hydrophobic amino acid, may be substituted by acodon encoding another less hydrophobic residue, such as glycine, or amore hydrophobic residue, such as valine, leucine, or isoleucine.Similarly, changes which result in substitution of one negativelycharged residue for another, such as aspartic acid for glutamic acid, orone positively charged residue for another, such as lysine for arginine,can also be expected to produce a functionally equivalent product.Nucleotide changes which result in alteration of the N-terminal andC-terminal portions of the polypeptide molecule would also not beexpected to alter the activity of the polypeptide. Each of the proposedmodifications is well within the routine skill in the art, as isdetermination of retention of biological activity of the encodedproducts. Consequently, an isolated polynucleotide comprising anucleotide sequence of at least 30 (preferably at least 40, mostpreferably at least 60) contiguous nucleotides derived from a nucleotidesequence selected from the group consisting of SEQ ID NO:1, 3, 5, 7, 9,11, 13, 15, 17, 19, 21, 23, 25, 27, or 29, and the complement of suchnucleotide sequences may be used to affect the expression and/orfunction of a LEC1-related transcription factor in a host cell. A methodof using an isolated polynucleotide to affect the level of expression ofa polypeptide in a host cell (eukaryotic, such as plant or yeast,prokaryotic such as bacterial) may comprise the steps of: constructingan isolated polynucleotide of the present invention or an isolatedchimeric gene of the present invention; introducing the isolatedpolynucleotide or the isolated chimeric gene into a host cell; measuringthe level of a polypeptide or enzyme activity in the host cellcontaining the isolated polynucleotide; and comparing the level of apolypeptide or enzyme activity in the host cell containing the isolatedpolynucleotide with the level of a polypeptide or enzyme activity in ahost cell that does not contain the isolated polynucleotide.

Moreover, substantially similar nucleic acid fragments may also becharacterized by their ability to hybridize. Estimates of such homologyare provided by either DNA-DNA or DNA-RNA hybridization under conditionsof stringency as is well understood by those skilled in the art (Hamesand Higgins, Eds. (1985) Nucleic Acid Hybridisation, IRL Press, Oxford,U.K.). Stringency conditions can be adjusted to screen for moderatelysimilar fragments, such as homologous sequences from distantly relatedorganisms, to highly similar fragments, such as genes that duplicatefunctional enzymes from closely related organisms. Post-hybridizationwashes determine stringency conditions. One set of preferred conditionsuses a series of washes starting with 6×SSC, 0.5% SDS at roomtemperature for 15 min, then repeated with 2×SSC, 0.5% SDS at 45° C. for30 min, and then repeated twice with 0.2×SSC, 0.5% SDS at 50° C. for 30min. A more preferred set of stringent conditions uses highertemperatures in which the washes are identical to those above except forthe temperature of the final two 30 min washes in 0.2×SSC, 0.5% SDS wasincreased to 60° C. Another preferred set of highly stringent conditionsuses two final washes in 0.1×SSC, 0.1% SDS at 65° C.

Substantially similar nucleic acid fragments of the instant inventionmay also be characterized by the percent identity of the amino acidsequences that they encode to the amino acid sequences disclosed herein,as determined by algorithms commonly employed by those skilled in thisart. Suitable nucleic acid fragments (isolated polynucleotides of thepresent invention) encode polypeptides that are at least about 70%identical, preferably at least about 80% identical to the amino acidsequences reported herein. Preferred nucleic acid fragments encode aminoacid sequences that are at least about 85% identical to the amino acidsequences reported herein. More preferred nucleic acid fragments encodeamino acid sequences that are at least about 90% identical to the aminoacid sequences reported herein. Most preferred are nucleic acidfragments that encode amino acid sequences that are at least about 95%identical to the amino acid sequences reported herein. Suitable nucleicacid fragments not only have the above identities but typically encode apolypeptide having at least 50 amino acids, preferably at least 100amino acids, more preferably at least 150 amino acids, still morepreferably at least 200 amino acids, and most preferably at least 250amino acids. Sequence alignments and percent identity calculations wereperformed using the Megalign program of the LASERGENE bioinformaticscomputing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of thesequences was performed using the Clustal method of alignment (Higginsand Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAPPENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwisealignments using the Clustal method were KTUPLE 1, GAP PENALTY=3,WINDOW=5 and DIAGONALS SAVED=5.

A “substantial portion” of an amino acid or nucleotide sequencecomprises an amino acid or a nucleotide sequence that is sufficient toafford putative identification of the protein or gene that the aminoacid or nucleotide sequence comprises. Amino acid and nucleotidesequences can be evaluated either manually by one skilled in the art, orby using computer-based sequence comparison and identification toolsthat employ algorithms such as BLAST (Basic Local Alignment Search Tool;Altschul et al. (1993) J. Mol. Biol. 215:403-410; see also theexplanation of the BLAST algorithm on the world wide web site for theNational Center for Biotechnology Information at the National Library ofMedicine of the National Institutes of Health). In general, a sequenceof ten or more contiguous amino acids or thirty or more contiguousnucleotides is necessary to putatively identify a polypeptide or nucleicacid sequence as homologous to a known protein or gene. Moreover, withrespect to nucleotide sequences, gene-specific oligonucleotide probescomprising 30 or more contiguous nucleotides may be used insequence-dependent methods of gene identification (e.g., Southernhybridization) and isolation (e.g., in situ hybridization of bacterialcolonies or bacteriophage plaques). In addition, short oligonucleotidesof 12 or more nucleotides may be used as amplification primers in PCR inorder to obtain a particular nucleic acid fragment comprising theprimers. Accordingly, a “substantial portion” of a nucleotide sequencecomprises a nucleotide sequence that will afford specific identificationand/or isolation of a nucleic acid fragment comprising the sequence. Theinstant specification teaches amino acid and nucleotide sequencesencoding polypeptides that comprise one or more particular plantproteins. The skilled artisan, having the benefit of the sequences asreported herein, may now use all or a substantial portion of thedisclosed sequences for purposes known to those skilled in this art.Accordingly, the instant invention comprises the complete sequences asreported in the accompanying Sequence Listing, as well as substantialportions of those sequences as defined above.

“Codon degeneracy” refers to divergence in the genetic code permittingvariation of the nucleotide sequence without effecting the amino acidsequence of an encoded polypeptide. Accordingly, the instant inventionrelates to any nucleic acid fragment comprising a nucleotide sequencethat encodes all or a substantial portion of the amino acid sequencesset forth herein. The skilled artisan is well aware of the “codon-bias”exhibited by a specific host cell in usage of nucleotide codons tospecify a given amino acid. Therefore, when synthesizing a nucleic acidfragment for improved expression in a host cell, it is desirable todesign the nucleic acid fragment such that its frequency of codon usageapproaches the frequency of preferred codon usage of the host cell.

“Synthetic nucleic acid fragments” can be assembled from oligonucleotidebuilding blocks that are chemically synthesized using procedures knownto those skilled in the art. These building blocks are ligated andannealed to form larger nucleic acid fragments that may then beenzymatically assembled to construct the entire desired nucleic acidfragment. “Chemically synthesized”, as related to a nucleic acidfragment, means that the component nucleotides were assembled in vitro.Manual chemical synthesis of nucleic acid fragments may be accomplishedusing well established procedures, or automated chemical synthesis canbe performed using one of a number of commercially available machines.Accordingly, the nucleic acid fragments can be tailored for optimal geneexpression based on optimization of the nucleotide sequence to reflectthe codon bias of the host cell. The skilled artisan appreciates thelikelihood of successful gene expression if codon usage is biasedtowards those codons favored by the host. Determination of preferredcodons can be based on a survey of genes derived from the host cellwhere sequence information is available.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, including regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence.“Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign-gene” refers to a genenot normally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

“Coding sequence” refers to a nucleotide sequence that codes for aspecific amino acid sequence. “Regulatory sequences” refer to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may includepromoters, translation leader sequences, introns, and polyadenylationrecognition sequences.

“Promoter” refers to a nucleotide sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. The promoter sequenceconsists of proximal and more distal upstream elements, the latterelements often referred to as enhancers. Accordingly, an “enhancer” is anucleotide sequence that can stimulate promoter activity and may be aninnate element of the promoter or a heterologous element inserted toenhance the level or tissue-specificity of a promoter. Promoters may bederived in their entirety from a native gene, or may be composed ofdifferent elements derived from different promoters found in nature, ormay even comprise synthetic nucleotide segments. It is understood bythose skilled in the art that different promoters may direct theexpression of a gene in different tissues or cell types, or at differentstages of development, or in response to different environmentalconditions. Promoters that cause a nucleic acid fragment to be expressedin most cell types at most times are commonly referred to as“constitutive promoters”. New promoters of various types useful in plantcells are constantly being discovered; numerous examples may be found inthe compilation by Okamuro and Goldberg (1989) Biochemistry of Plants15:1-82. It is further recognized that since in most cases the exactboundaries of regulatory sequences have not been completely defined,nucleic acid fragments of different lengths may have identical promoteractivity.

“Translation leader sequence” refers to a nucleotide sequence locatedbetween the promoter sequence of a gene and the coding sequence. Thetranslation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency. Examples of translation leadersequences have been described (Turner and Foster (1995) Mol. Biotechnol.3:225-236).

“3′ non-coding sequences” refer to nucleotide sequences locateddownstream of a coding sequence and include polyadenylation recognitionsequences and other sequences encoding regulatory signals capable ofaffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor. The use of different 3′non-coding sequences is exemplified by Ingelbrecht et al. (1989) PlantCell 1:671-680.

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from posttranscriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated intopolypeptides by the cell. “cDNA” refers to DNA that is complementary toand derived from an mRNA template. The cDNA can be single-stranded orconverted to double stranded form using, for example, the Klenowfragment of DNA polymerase I. “Sense-RNA” refers to an RNA transcriptthat includes the mRNA and so can be translated into a polypeptide bythe cell. “Antisense RNA” refers to an RNA transcript that iscomplementary to all or part of a target primary transcript or mRNA andthat blocks the expression of a target gene (see U.S. Pat. No.5,107,065, incorporated herein by reference). The complementarity of anantisense RNA may be with any part of the specific nucleotide sequence,i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, orthe coding sequence. “Functional RNA” refers to sense RNA, antisenseRNA, ribozyme RNA, or other RNA that may not be translated but yet hasan effect on cellular processes.

The term “operably linked” refers to the association of two or morenucleic acid fragments on a single polynucleotide so that the functionof one is affected by the other. For example, a promoter is operablylinked with a coding sequence when it is capable of affecting theexpression of that coding sequence (i.e., that the coding sequence isunder the transcriptional control of the promoter). Coding sequences canbe operably linked to regulatory sequences in sense or antisenseorientation.

The term “expression”, as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid fragment of the invention. Expression may also refer totranslation of mRNA into a polypeptide. “Antisense inhibition” refers tothe production of antisense RNA transcripts capable of suppressing theexpression of the target protein. “Overexpression” refers to theproduction of a gene product in transgenic organisms that exceeds levelsof production in normal or non-transformed organisms. “Co-suppression”refers to the production of sense RNA transcripts capable of suppressingthe expression of identical or substantially similar foreign orendogenous genes (U.S. Pat. No. 5,231,020, incorporated herein byreference).

A “protein” or “polypeptide” is a chain of amino acids arranged in aspecific order determined by the coding sequence in a polynucleotideencoding the polypeptide. Each protein or polypeptide has a uniquefunction.

“Altered levels” or “altered expression” refers to the production ofgene product(s) in transgenic organisms in amounts or proportions thatdiffer from that of normal or non-transformed organisms.

“Mature protein” or the term “mature” when used in describing a proteinrefers to a post-translationally processed polypeptide; i.e., one fromwhich any pre- or propeptides present in the primary translation producthave been removed. “Precursor protein” or the term “precursor” when usedin describing a protein refers to the primary product of translation ofmRNA; i.e., with pre- and propeptides still present. Pre- andpropeptides may be but are not limited to intracellular localizationsignals.

A “chloroplast transit peptide” is an amino acid sequence which istranslated in conjunction with a protein and directs the protein to thechloroplast or other plastid types present in the cell in which theprotein is made. “Chloroplast transit sequence” refers to a nucleotidesequence that encodes a chloroplast transit peptide. A “signal peptide”is an amino acid sequence that is translated in conjunction with aprotein and directs the protein to the secretory system (Chrispeels(1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53). If the proteinis to be directed to a vacuole, a vacuolar targeting signal (supra) canfurther be added, or if to the endoplasmic reticulum, an endoplasmicreticulum retention signal (supra) may be added. If the protein is to bedirected to the nucleus, any signal peptide present should be removedand instead a nuclear localization signal included (Raikhel (1992) PlantPhys. 100:1627-1632).

“Transformation” refers to the transfer of a nucleic acid fragment intothe genome of a host organism, resulting in genetically stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” organisms. Examples of methodsof plant transformation include Agrobacterium-mediated transformation(De Blaere et al. (1987) Meth. Enzymol. 143:277) andparticle-accelerated or “gene gun” transformation technology (Klein etal. (1987) Nature (London) 327:70-73; U.S. Pat. No. 4,945,050,incorporated herein by reference). Thus, isolated polynucleotides of thepresent invention can be incorporated into recombinant constructs,typically DNA constructs, capable of introduction into and replicationin a host cell. Such a construct can be a vector that includes areplication system and sequences that are capable of transcription andtranslation of a polypeptide-encoding sequence in a given host cell. Anumber of vectors suitable for stable transfection of plant cells or forthe establishment of transgenic plants have been described in, e.g.,Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985, supp. 1987;Weissbach and Weissbach, Methods for Plant Molecular Biology, AcademicPress, 1989; and Flevin et al., Plant Molecular Biology Manual, KluwerAcademic Publishers, 1990. Typically, plant expression vectors include,for example, one or more cloned plant genes under the transcriptionalcontrol of 5′ and 3′ regulatory sequences and a dominant selectablemarker. Such plant expression vectors also can contain a promoterregulatory region (e.g., a regulatory region controlling inducible orconstitutive, environmentally- or developmentally-regulated, or cell- ortissue-specific expression), a transcription initiation start site, aribosome binding site, an RNA processing signal, a transcriptiontermination site, and/or a polyadenylation signal.

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook etal. Molecular Cloning: A Laboratory Manual; Cold Spring HarborLaboratory Press: Cold Spring Harbor, 1989 (hereinafter “Maniatis”).

“PCR” or “polymerase chain reaction” is well known, by those skilled inthe art, as a technique that is used for the amplification of specificDNA segments (U.S. Pat. Nos. 4,683,195 and 4,800,159).

The present invention concerns an isolated polynucleotide comprising anucleotide sequence encoding a LEC1-related transcription factorpolypeptide having at least 90% identity, based on the Clustal method ofalignment, when compared to a polypeptide selected from the groupconsisting of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28 or 30.

This invention also relates to the isolated complement of suchpolynucleotides, wherein the complement and the polynucleotide consistof the same number of nucleotides, and the nucleotide sequences of thecomplement and the polynucleotide have 100% complementarity.

Nucleic acid fragments encoding at least a portion of severalLEC1-related transcription factors have been isolated and identified bycomparison of random plant cDNA sequences to public databases containingnucleotide and protein sequences using the BLAST algorithms well knownto those skilled in the art. The nucleic acid fragments of the instantinvention may be used to isolate cDNAs and genes encoding homologousproteins from the same or other plant species. Isolation of homologousgenes using sequence-dependent protocols is well known in the art.Examples of sequence-dependent protocols include, but are not limitedto, methods of nucleic acid hybridization, and methods of DNA and RNAamplification as exemplified by various uses of nucleic acidamplification technologies (e.g., polymerase chain reaction, ligasechain reaction).

For example, genes encoding other LEC1-related transcription factors,either as cDNAs or genomic DNAs, could be isolated directly by using allor a portion of the instant nucleic acid fragments as DNA hybridizationprobes to screen libraries from any desired plant employing methodologywell known to those skilled in the art. Specific oligonucleotide probesbased upon the instant nucleic acid sequences can be designed andsynthesized by methods known in the art (Maniatis). Moreover, an entiresequence can be used directly to synthesize DNA probes by methods knownto the skilled artisan such as random primer DNA labeling, nicktranslation, end-labeling techniques, or RNA probes using available invitro transcription systems. In addition, specific primers can bedesigned and used to amplify a part or all of the instant sequences. Theresulting amplification products can be labeled directly duringamplification reactions or labeled after amplification reactions, andused as probes to isolate full length cDNA or genomic fragments underconditions of appropriate stringency.

In addition, two short segments of the instant nucleic acid fragmentsmay be used in polymerase chain reaction protocols to amplify longernucleic acid fragments encoding homologous genes from DNA or RNA. Thepolymerase chain reaction may also be performed on a library of clonednucleic acid fragments wherein the sequence of one primer is derivedfrom the instant nucleic acid fragments, and the sequence of the otherprimer takes advantage of the presence of the polyadenylic acid tractsto the 3′ end of the mRNA precursor encoding plant genes. Alternatively,the second primer sequence may be based upon sequences derived from thecloning vector. For example, the skilled artisan can follow the RACEprotocol (Frohman et al. (1988) Proc. Natl. Acad. Sci. USA 85:8998-9002)to generate cDNAs by using PCR to amplify copies of the region between asingle point in the transcript and the 3′ or 5′ end. Primers oriented inthe 3′ and 5′ directions can be designed from the instant sequences.Using commercially available 3′ RACE or 5′ RACE systems (BRL), specific3′ or 5′ cDNA fragments can be isolated (Ohara et al. (1989) Proc. Natl.Acad. Sci. USA 86:5673-5677; Loh et al. (1989) Science 243:217-220).Products generated by the 3′ and 5′ RACE procedures can be combined togenerate full-length cDNAs (Frohman and Martin (1989) Techniques 1:165).Consequently, a polynucleotide comprising a nucleotide sequence of atleast 30 (preferably at least 40, most preferably at least 60)contiguous nucleotides derived from a nucleotide sequence selected fromthe group consisting of SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,23, 25, 27, or 29 and the complement of such nucleotide sequences may beused in such methods to obtain a nucleic acid fragment encoding asubstantial portion of an amino acid sequence of a polypeptide.

Availability of the instant nucleotide and deduced amino acid sequencesfacilitates immunological screening of cDNA expression libraries.Synthetic peptides representing portions of the instant amino acidsequences may be synthesized. These peptides can be used to immunizeanimals to produce polyclonal or monoclonal antibodies with specificityfor peptides or proteins comprising the amino acid sequences. Theseantibodies can be then be used to screen cDNA expression libraries toisolate full-length cDNA clones of interest (Lerner (1984) Adv. Immunol.36:1-34; Maniatis).

In another embodiment, this invention concerns viruses and host cellscomprising either the chimeric genes of the invention as describedherein or an isolated polynucleotide of the invention as describedherein. Examples of host cells that can be used to practice theinvention include, but are not limited to, yeast, bacteria, and plants.

As was noted above, the nucleic acid fragments of the instant inventionmay be used to create transgenic plants in which the disclosedpolypeptides are present at higher levels when overexpressed, or lowerlevels when cosuppressed, than normal levels, or in cell types ordevelopmental stages in which they are not normally found. This wouldhave the effect of altering the level of gene expression for aLEC1-related transcription factor, which in turn would lead to alteredexpression of those genes controlled by the Lec1-related transcriptionfactor. This would lead to developmental and phenotypic variations, suchas but not limited to, over-accumulation of oils in tissues of theplant. For example, a seed specific promoter directing theoverexpression of a Lec-1 related transcription factor will lead to theoverexpression of oil in the seed.

Overexpression of the proteins of the instant invention may beaccomplished by first constructing a chimeric gene in which the codingregion is operably linked to a promoter capable of directing expressionof a gene in the desired tissues at the desired stage of development.The chimeric gene may comprise promoter sequences and translation leadersequences derived from the same genes. 3′ Non-coding sequences encodingtranscription termination signals may also be provided. The instantchimeric gene may also comprise one or more introns in order tofacilitate gene expression.

Plasmid vectors comprising the instant isolated polynucleotide (orchimeric gene) may be constructed. The choice of plasmid vector isdependent upon the method that will be used to transform host plants.The skilled artisan is well aware of the genetic elements that must bepresent on the plasmid vector in order to successfully transform, selectand propagate host cells containing the chimeric gene. The skilledartisan will also recognize that different independent transformationevents will result in different levels and patterns of expression (Joneset al. (1985) EMBO J. 4:2411-2418; De Almeida et al. (1989) Mol. Gen.Genetics 218:78-86), and thus that multiple events must be screened inorder to obtain lines displaying the desired expression level andpattern. Such screening may be accomplished by Southern analysis of DNA,Northern analysis of mRNA expression, Western analysis of proteinexpression, or phenotypic analysis.

For some applications it may be useful to direct the instantpolypeptides to different cellular compartments, or to facilitate itssecretion from the cell. It is thus envisioned that the chimeric genedescribed above may be further supplemented by directing the codingsequence to encode the instant polypeptides with appropriateintracellular targeting sequences such as transit sequences (Keegstra(1989) Cell 56:247-253), signal sequences or sequences encodingendoplasmic reticulum localization (Chrispeels (1991) Ann. Rev. PlantPhys. Plant Mol. Biol. 42:21-53), or nuclear localization signals(Raikhel (1992) Plant Phys. 100:1627-1632) with or without removingtargeting sequences that are already present. While the references citedgive examples of each of these, the list is not exhaustive and moretargeting signals of use may be discovered in the future.

It may also be desirable to reduce or eliminate expression of genesencoding the instant polypeptides in plants for some applications. Inorder to accomplish this, a chimeric gene designed for co-suppression ofthe instant polypeptide can be constructed by linking a gene or genefragment encoding that polypeptide to plant promoter sequences.Alternatively, a chimeric gene designed to express antisense RNA for allor part of the instant nucleic acid fragment can be constructed bylinking the gene or gene fragment in reverse orientation to plantpromoter sequences. Either the co-suppression or antisense chimericgenes could be introduced into plants via transformation whereinexpression of the corresponding endogenous genes are reduced oreliminated.

Molecular genetic solutions to the generation of plants with alteredgene expression have a decided advantage over more traditional plantbreeding approaches. Changes in plant phenotypes can be produced byspecifically inhibiting expression of one or more genes by antisenseinhibition or cosuppression (U.S. Pat. Nos. 5,190,931, 5,107,065 and5,283,323). An antisense or cosuppression construct would act as adominant negative regulator of gene activity. While conventionalmutations can yield negative regulation of gene activity these effectsare most likely recessive. The dominant negative regulation availablewith a transgenic approach may be advantageous from a breedingperspective. In addition, the ability to restrict the expression of aspecific phenotype to the reproductive tissues of the plant by the useof tissue specific promoters may confer agronomic advantages relative toconventional mutations that may have an effect in all tissues in which amutant gene is ordinarily expressed.

The person skilled in the art will know that special considerations areassociated with the use of antisense or cosuppression technologies inorder to reduce expression of particular genes. For example, the properlevel of expression of sense or antisense genes may require the use ofdifferent chimeric genes utilizing different regulatory elements knownto the skilled artisan. Once transgenic plants are obtained by one ofthe methods described above, it will be necessary to screen individualtransgenics for those that most effectively display the desiredphenotype. Accordingly, the skilled artisan will develop methods forscreening large numbers of transformants. The nature of these screenswill generally be chosen on practical grounds. For example, one canscreen by looking for changes in gene expression by using antibodiesspecific for the protein encoded by the gene being suppressed, or onecould establish assays that specifically measure enzyme activity. Apreferred method will be one that allows large numbers of samples to beprocessed rapidly, since it will be expected that a large number oftransformants will be negative for the desired phenotype.

In another embodiment, the present invention concerns a LEC1-relatedtranscription factor polypeptide having an amino acid sequence that isat least 90% identical, based on the Clustal method of alignment, to apolypeptide selected from the group consisting of SEQ ID NO:2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30.

The instant polypeptides (or portions thereof) may be produced inheterologous host cells, particularly in the cells of microbial hosts,and can be used to prepare antibodies to these proteins by methods wellknown to those skilled in the art. The antibodies are useful fordetecting the polypeptides of the instant invention in situ in cells orin vitro in cell extracts. Preferred heterologous host cells forproduction of the instant polypeptides are microbial hosts. Microbialexpression systems and expression vectors containing regulatorysequences that direct high level expression of foreign proteins are wellknown to those skilled in the art. Any of these could be used toconstruct a chimeric gene for production of the instant polypeptides.This chimeric gene could then be introduced into appropriatemicroorganisms via transformation to provide high level expression ofthe encoded LEC 1-related transcription factor. An example of a vectorfor high level expression of the instant polypeptides in a bacterialhost is provided (Example 7).

All or a substantial portion of the polynucleotides of the instantinvention may also be used as probes for genetically and physicallymapping the genes that they are a part of, and used as markers fortraits linked to those genes. Such information may be useful in plantbreeding in order to develop lines with desired phenotypes. For example,the instant nucleic acid fragments may be used as restriction fragmentlength polymorphism (RFLP) markers. Southern blots (Maniatis) ofrestriction-digested plant genomic DNA may be probed with the nucleicacid fragments of the instant invention. The resulting banding patternsmay then be subjected to genetic analyses using computer programs suchas MapMaker (Lander et al. (1987) Genomics 1:174-181) in order toconstruct a genetic map. In addition, the nucleic acid fragments of theinstant invention may be used to probe Southern blots containingrestriction endonuclease-treated genomic DNAs of a set of individuals,representing parent and progeny of a defined genetic cross. Segregationof the DNA polymorphisms is noted and used to calculate the position ofthe instant nucleic acid sequence in the genetic map previously obtainedusing this population (Botstein et al. (1980) Am. J. Hum. Genet.32:314-331).

The production and use of plant gene-derived probes for use in geneticmapping is described in Bernatzky and Tanksley (1986) Plant Mol. Biol.Reporter 4:37-41. Numerous publications describe genetic mapping ofspecific cDNA clones using the methodology outlined above or variationsthereof. For example, F2 intercross populations, backcross populations,randomly mated populations, near isogenic lines, and other sets ofindividuals may be used for mapping. Such methodologies are well knownto those skilled in the art.

Nucleic acid probes derived from the instant nucleic acid sequences mayalso be used for physical mapping (i.e., placement of sequences onphysical maps; see Hoheisel et al. In: Nonmammalian Genomic Analysis: APractical Guide, Academic press 1996, pp. 319-346, and references citedtherein).

Nucleic acid probes derived from the instant nucleic acid sequences maybe used in direct fluorescence in situ hybridization (FISH) mapping(Trask (1991) Trends Genet. 7:149-154). Although current methods of FISHmapping favor use of large clones (several to several hundred KB; seeLaan et al. (1995) Genome Res. 5:13-20), improvements in sensitivity mayallow performance of FISH mapping using shorter probes.

A variety of nucleic acid amplification-based methods of genetic andphysical mapping may be carried out using the instant nucleic acidsequences. Examples include allele-specific amplification (Kazazian(1989) J. Lab. Clin. Med. 11:95-96), polymorphism of PCR-amplifiedfragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332),allele-specific ligation (Landegren et al. (1988) Science241:1077-1080), nucleotide extension reactions (Sokolov (1990) NucleicAcid Res. 18:3671), Radiation Hybrid Mapping (Walter et al. (1997) Nat.Genet. 7:22-28) and Happy Mapping (Dear and Cook (1989) Nucleic AcidRes. 17:6795-6807). For these methods, the sequence of a nucleic acidfragment is used to design and produce primer pairs for use in theamplification reaction or in primer extension reactions. The design ofsuch primers is well known to those skilled in the art. In methodsemploying PCR-based genetic mapping, it may be necessary to identify DNAsequence differences between the parents of the mapping cross in theregion corresponding to the instant nucleic acid sequence. This,however, is generally not necessary for mapping methods.

Loss of function mutant phenotypes may be identified for the instantcDNA clones either by targeted gene disruption protocols or byidentifying specific mutants for these genes contained in a maizepopulation carrying mutations in all possible genes (Ballinger andBenzer (1989) Proc. Natl. Acad. Sci. USA 86:9402-9406; Koes et al.(1995) Proc. Natl. Acad. Sci. USA 92:8149-8153; Bensen et al. (1995)Plant Cell 7:75-84). The latter approach may be accomplished in twoways. First, short segments of the instant nucleic acid fragments may beused in polymerase chain reaction protocols in conjunction with amutation tag sequence primer on DNAs prepared from a population ofplants in which Mutator transposons or some other mutation-causing DNAelement has been introduced (see Bensen, supra). The amplification of aspecific DNA fragment with these primers indicates the insertion of themutation tag element in or near the plant gene encoding the instantpolypeptides. Alternatively, the instant nucleic acid fragment may beused as a hybridization probe against PCR amplification productsgenerated from the mutation population using the mutation tag sequenceprimer in conjunction with an arbitrary genomic site primer, such asthat for a restriction enzyme site-anchored synthetic adaptor. Witheither method, a plant containing a mutation in the endogenous geneencoding the instant polypeptides can be identified and obtained. Thismutant plant can then be used to determine or confirm the naturalfunction of the instant polypeptides disclosed herein.

EXAMPLES

The present invention is further defined in the following Examples, inwhich parts and percentages are by weight and degrees are Celsius,unless otherwise stated. It should be understood that these Examples,while indicating preferred embodiments of the invention, are given byway of illustration only. From the above discussion and these Examples,one skilled in the art can ascertain the essential characteristics ofthis invention, and without departing from the spirit and scope thereof,can make various changes and modifications of the invention to adapt itto various usages and conditions. Thus, various modifications of theinvention in addition to those shown and described herein will beapparent to those skilled in the art from the foregoing description.Such modifications are also intended to fall within the scope of theappended claims.

The disclosure of each reference set forth herein is incorporated hereinby reference in its entirety.

Example 1 Composition of cDNA Libraries; Isolation and Sequencing ofcDNA Clones

cDNA libraries representing mRNAs from Momordica charantia, Eucalyptustereticomis, corn, rice, soybean, wheat, and Canna edulis tissues wereprepared. The characteristics of the libraries are described below.

TABLE 2 cDNA Libraries from Momordica charantia, Eucalyptustereticornis, Corn, Rice, Soybean, Wheat, and Canna edulis LibraryTissue Clone fds Momordica charantia developing seed fds.pk0003.h5 ect1cCanna edulis tubers ect1c.pk007.p18 eef1c Eucalyptus tereticornis flowerbuds from adult tree eef1c.pk004.c8 cbn10 Corn developing kernel (embryoand endosperm); 10 cbn10.pk0005.e6 days after pollination p0006 Cornyoung shoot p0006.cbysa51r rl0n Rice 15 day old leaf* rl0n.pk0061.c8rsl1n Rice 15-day-old seedling* rsl1n.pk002.g10 ses4d Soybeanembryogenic suspension 4 days after ses4d.pk0037.e3 subculture src2cSoybean 8 day old root infected with cyst nematode src2c.pk003.i13Heterodera glycenis src2c.pk011.m12 src2c.pk025.b3 src3c Soybean 8 dayold root infected with cyst nematode src3c.pk028.j21 Heterodera glyceniswkm1c Wheat kernel malted 55 hours at 22 degrees celsius wkm1c.pk0002.d7wlk8 Wheat seedlings 8 hours after treatment with wlk8.pk0001.e10herbicide** wlm96 Wheat seedlings 96 hours after inoculation withwlm96.pk037.k9 Erysiphe graminis f. sp tritici *These libraries werenormalized essentially as described in U.S. Pat. No. 5,482,845,incorporated herein by reference. **Application of6-iodo-2-propoxy-3-propyl-4(3H)-quinazolinone; synthesis and methods ofusing this compound are described in USSN 08/545,827, incorporatedherein by reference.

cDNA libraries may be prepared by any one of many methods available. Forexample, the cDNAs may be introduced into plasmid vectors by firstpreparing the cDNA libraries in Uni-ZAP™ XR vectors according to themanufacturer's protocol (Stratagene Cloning Systems, La Jolla, Calif.).The Uni-ZAP™ XR libraries are converted into plasmid libraries accordingto the protocol provided by Stratagene. Upon conversion, cDNA insertswill be contained in the plasmid vector pBluescript. In addition, thecDNAs may be introduced directly into precut Bluescript II SK(+) vectors(Stratagene) using T4 DNA ligase (New England Biolabs), followed bytransfection into DH10B cells according to the manufacturer's protocol(GIBCO BRL Products). Once the cDNA inserts are in plasmid vectors,plasmid DNAs are prepared from randomly picked bacterial coloniescontaining recombinant pBluescript plasmids, or the insert cDNAsequences are amplified via polymerase chain reaction using primersspecific for vector sequences flanking the inserted cDNA sequences.Amplified insert DNAs or plasmid DNAs are sequenced in dye-primersequencing reactions to generate partial cDNA sequences. (expressedsequence tags or “ESTs”; see Adams et al., (1991) Science252:1651-1656). The resulting ESTs are analyzed using a Perkin ElmerModel 377 fluorescent sequencer.

Full-insert sequence (FIS) data is generated utilizing a modifiedtransposition protocol. Clones identified for FIS are recovered fromarchived glycerol stocks as single colonies, and plasmid DNAs areisolated via alkaline lysis. Isolated DNA templates are reacted withvector primed M13 forward and reverse oligonucleotides in a PCR-basedsequencing reaction and loaded onto automated sequencers. Confirmationof clone identification is performed by sequence alignment to theoriginal EST sequence from which the FIS request is made.

Confirmed templates are transposed via the Primer Island transpositionkit (PE Applied Biosystems, Foster City, Calif.) which is based upon theSaccharomyces cerevisiae Ty1 transposable element (Devine and Boeke(1994) Nucleic Acids Res. 22:3765-3772). The in vitro transpositionsystem places unique binding sites randomly throughout a population oflarge DNA molecules. The transposed DNA is then used to transform DH10Belectro-competent cells (Gibco BRL/Life Technologies, Rockville, Md.)via electroporation. The transposable element contains an additionalselectable marker (named DHFR; Fling and Richards (1983) Nucleic AcidsRes. 11:5147-5158), allowing for dual selection on agar plates of onlythose subclones containing the integrated transposon. Multiple subclonesare randomly selected from each transposition reaction, plasmid DNAs areprepared via alkaline lysis, and templates are sequenced (ABI Prismdye-terminator ReadyReaction mix) outward from the transposition eventsite, utilizing unique primers specific to the binding sites within thetransposon.

Sequence data is collected (ABI Prism Collections) and assembled usingPhred/Phrap (P. Green, University of Washington, Seattle). Phred/Phrapis a public domain software program which re-reads the ABI sequencedata, re-calls the bases, assigns quality values, and writes the basecalls and quality values into editable output files. The Phrap sequenceassembly program uses these quality values to increase the accuracy ofthe assembled sequence contigs. Assemblies are viewed by the Consedsequence editor (D. Gordon, University of Washington, Seattle).

In some of the clones the cDNA fragment corresponds to a portion of the3′-terminus of the gene and does not cover the entire open readingframe. In order to obtain the upstream information one of two differentprotocols are used. The first of these methods results in the productionof a fragment of DNA containing a portion of the desired gene sequencewhile the second method results in the production of a fragmentcontaining the entire open reading frame. Both of these methods use tworounds of PCR amplification to obtain fragments from one or morelibraries. The libraries some times are chosen based on previousknowledge that the specific gene should be found in a certain tissue andsome times are randomly-chosen. Reactions to obtain the same gene may beperformed on several libraries in parallel or on a pool of libraries.Library pools are normally prepared using from 3 to 5 differentlibraries and normalized to a uniform dilution. In the first round ofamplification both methods use a vector-specific (forward) primercorresponding to a portion of the vector located at the 5′-terminus ofthe clone coupled with a gene-specific (reverse) primer. The firstmethod uses a sequence that is complementary to a portion of the alreadyknown gene sequence while the second method uses a gene-specific primercomplementary to a portion of the 3′-untranslated region (also referredto as UTR). In the second round of amplification a nested set of primersis used for both methods. The resulting DNA fragment is ligated into apBluescript vector using a commercial kit and following themanufacturer's protocol. This kit is selected from many available fromseveral vendors including Invitrogen (Carlsbad, Calif.), Promega Biotech(Madison, Wis.), and Gibco-BRL (Gaithersburg, Md.). The plasmid DNA isisolated by alkaline lysis method and submitted for sequencing andassembly using Phred/Phrap, as above.

Example 2 Identification of cDNA Clones

cDNA clones encoding LEC1-related transcription factors were identifiedby conducting BLAST (Basic Local Alignment Search Tool; Altschul et al.(1993) J. Mol. Biol. 215:403-410; see also the explanation of the BLASTalgorithm on the world wide web site for the National Center forBiotechnology Information at the National Library of Medicine of theNational Institutes of Health) searches for similarity to sequencescontained in the BLAST “nr” database (comprising all non-redundantGenBank CDS translations, sequences derived from the 3-dimensionalstructure Brookhaven Protein Data Bank, the last major release of theSWISS-PROT protein sequence database, EMBL, and DDBJ databases). ThecDNA sequences obtained in Example 1 were analyzed for similarity to allpublicly available DNA sequences contained in the “nr” database usingthe BLASTN algorithm provided by the National Center for BiotechnologyInformation (NCBI). The DNA sequences were translated in all readingframes and compared for similarity to all publicly available proteinsequences contained in the “nr” database using the BLASTX algorithm(Gish and States (1993) Nat. Genet. 3:266-272) provided by the NCBI. Forconvenience, the P-value (probability) of observing a match of a cDNAsequence to a sequence contained in the searched databases merely bychance as calculated by BLAST are reported herein as “pLog” values,which represent the negative of the logarithm of the reported P-value.Accordingly, the greater the pLog value, the greater the likelihood thatthe cDNA sequence and the BLAST “hit” represent homologous proteins.

ESTs submitted for analysis are compared to the genbank database asdescribed above. ESTs that contain sequences more 5- or 3-prime can befound by using the BLASTn algorithm (Altschul et al (1997) Nucleic AcidsRes. 25:3389-3402.) against the Du Pont proprietary database comparingnucleotide sequences that share common or overlapping regions ofsequence homology. Where common or overlapping sequences exist betweentwo or more nucleic acid fragments, the sequences can be assembled intoa single contiguous nucleotide sequence, thus extending the originalfragment in either the 5 or 3 prime direction. Once the most 5-prime ESTis identified, its complete sequence can be determined by Full InsertSequencing as described in Example 1. Homologous genes belonging todifferent species can be found by comparing the amino acid sequence of aknown gene (from either a proprietary source or a public database)against an EST database using the tBLASTn algorithm. The tBLASTnalgorithm searches amino acid queries against a nucleotide database thatis translated in all 6 reading frames. This search allows fordifferences in nucleotide codon usage between different species, and forcodon degeneracy.

Example 3 Characterization of cDNA Clones Encoding LEC1-RelatedTranscription Factors

The BLASTX search using the EST sequences from clones listed in Table 3revealed similarity of the polypeptides encoded by the cDNAs toLEC-1-related transcription factor from Arabidopsis thaliana (NCBIGeneral Identifier No. gi 6729485), Arabidopsis thaliana (NCBI GeneralIdentifier No. gi 2398529), Arabidopsis thaliana (NCBI GeneralIdentifier No. gi 3738293), Zea mays (NCBI General Identifier No. gi22380). Shown in Table 3 are the BLAST results for individual ESTs(“EST”), the sequences of the entire cDNA inserts comprising theindicated cDNA clones (“FIS”), the sequences of contigs assembled fromtwo or more ESTs (“Contig”), sequences of contigs assembled from an FISand one or more ESTs (“Contig*”), or sequences encoding an entireprotein derived from an FIS, a contig, or an FIS and PCR (“CGS”):

TABLE 3 BLAST Results for Sequences Encoding Polypeptides Homologous toArabidopsis thaliana and Zea mays LEC-1-Related Transcription FactorsBLAST pLog Score Clone Status (NCBI General Identifier No.)fds.pk0003.h5 CGS 57.70 (gi 6729485) eef1c.pk004.c8 CGS 61.70 (gi 22380)cbn10.pk0005.e6 CGS 72.22 (gi 22380) p0006.cbysa51r CGS 55.52 (gi2244810) rl0n.pk0061.c8 CGS 46.52 (22380) rsl1n.pk002.g10 CGS 68.70 (gi22380) ses4d.pk0037.e3 CGS 49.00 (gi 2398529) src2c.pk003.i13 CGS 41.10(gi 3738293) src2c.pk011.m12 CGS 62.00 (gi 6729485) src2c.pk025.b3 CGS45.52 (gi 22380) src3c.pk028.j21 CGS 54.30 (gi 22380) wkm1c.pk0002.d7CGS 79.52 (gi 22380) wlk8.pk0001.e10 CGS 52.70 (gi 2398529)wlm96.pk037.k9 CGS 73.52 (gi 22380) ect1c.pk007.p18 FIS 44.70 (gi 22380)

The sequence of the entire cDNA insert in the clones listed in Table 3was determined. Further sequencing and searching of the DuPontproprietary database allowed the identification of other Momordica,Eucalyptus, Canna, corn, rice, soybean and/or wheat clones encodingLec1-related transcription factors. The BLASTX search using the ESTsequences from clones listed in Table 4 revealed similarity of thepolypeptides encoded by the cDNAs to Lec1-related transcription factorsfrom Arabidopsis thaliana (NCBI General Identifier No. 6729485, SEQ IDNO:31), Zea mays (NCBI General Identifier No. 22380, SEQ ID NO:32),Arabidopsis thaliana (NCBI General Identifier No. 2244810, SEQ IDNO:33), Arabidopsis thaliana (NCBI General Identifier No. 2398529, SEQID NO:34), Arabidopsis thaliana (NCBI General Identifier No. 3738293,SEQ ID NO:35). Shown in Table 4 are the BLAST results for individualESTs (“EST”), sequences of the entire cDNA inserts comprising theindicated cDNA clones (“FIS”), sequences of contigs assembled from twoor more ESTs (“Contig”), sequences of contigs assembled from an FIS andone or more ESTs (“Contig*”), or sequences derived from an FIS, acontig, or an FIS and PCR and encoding the entire protein (“CGS”):

TABLE 4 Percent Identity of Amino Acid Sequences Deduced From theNucleotide Sequences of cDNA Clones Encoding Polypeptides Homologous toArabidopsis thaliana and Zea mays LEC-1-Related Transcription FactorsPercent Identity to SEQ ID NO. (NCBI General Identifier No.) 2 68% (gi6729485) 4 62% (gi 22380) 6 80% (gi 22380) 8 48% (gi 2244810) 10 45% (gi22380) 12 81% (gi 22380) 14 47% (gi 2398529) 16 52% (gi 3738293) 18 73%(gi 6729485) 20 64% (gi 22380) 22 62% (gi 22380) 24 86% (gi 22380) 2654% (gi 2398529) 28 77% (gi 22380) 30 70% (gi 22380)

Alignments of the disclosed sequences are shown in FIG. 1. A core regionof approximately 90 amino acids is conserved in all of the polypeptidessequences. This region comprises a necessary functional domain for thetranscription factor. Sequence alignments and percent identitycalculations were performed using the Megalign program of the LASERGENEbioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiplealignment of the sequences was performed using the Clustal method ofalignment (Higgins and Sharp (1989) CABIOS. 5:151-153) with the defaultparameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parametersfor pairwise alignments using the Clustal method were KTUPLE 1, GAPPENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. Sequence alignments and BLASTscores and probabilities indicate that the nucleic acid fragmentscomprising the instant cDNA clones encode a substantial portion of aLEC-1-related transcription factor. These sequences represent the firstMorordica, Eucalyptus, rice, soybean and wheat and new corn sequencesencoding LEC-1-related transcription factors known to Applicant.

Example 5 Expression of Chimeric Genes in Monocot Cells

A chimeric gene comprising a cDNA encoding the instant polypeptides insense orientation with respect to the maize 27 kD zein promoter that islocated 5′ to the cDNA fragment, and the 10 kD zein 3′ end that islocated 3′ to the cDNA fragment, can be constructed. The cDNA fragmentof this gene may be generated by polymerase chain reaction (PCR) of thecDNA clone using appropriate oligonucleotide primers. Cloning sites(NcoI or SmaI) can be incorporated into the oligonucleotides to provideproper orientation of the DNA fragment when inserted into the digestedvector pML103 as described below. Amplification is then performed in astandard PCR. The amplified DNA is then digested with restrictionenzymes NcoI and SmaI and fractionated on an agarose gel. Theappropriate band can be isolated from the gel and combined with a 4.9 kbNcoI-SmaI fragment of the plasmid pML103. Plasmid pML103 has beendeposited under the terms of the Budapest Treaty at ATCC (American TypeCulture Collection, 10801 University Blvd., Manassas, Va. 20110-2209),and bears accession number ATCC 97366. The DNA segment from pML103contains a 1.05 kb SalI-NcoI promoter fragment of the maize 27 kD zeingene and a 0.96 kb SmaI-SalI fragment from the 3′ end of the maize 10 kDzein gene in the vector pGem9Zf(+) (Promega). Vector and insert DNA canbe ligated at 15° C. overnight, essentially as described (Maniatis). Theligated DNA may then be used to transform E. coli XL1-Blue (EpicurianColi XL-1 Blue™; Stratagene). Bacterial transformants can be screened byrestriction enzyme digestion of plasmid DNA and limited nucleotidesequence analysis using the dideoxy chain termination method (Sequenase™DNA Sequencing Kit; U.S. Biochemical). The resulting plasmid constructwould comprise a chimeric gene encoding, in the 5′ to 3′ direction, themaize 27 kD zein promoter, a cDNA fragment encoding the instantpolypeptides, and the 10 kD zein 3′ region.

The chimeric gene described above can then be introduced into corn cellsby the following procedure. Immature corn embryos can be dissected fromdeveloping caryopses derived from crosses of the inbred corn lines H99and LH132. The embryos are isolated 10 to 11 days after pollination whenthey are 1.0 to 1.5 mm long. The embryos are then placed with theaxis-side facing down and in contact with agarose-solidified N6 medium(Chu et al. (1975) Sci. Sin. Peking 18:659-668). The embryos are kept inthe dark at 27° C. Friable embryogenic callus consisting ofundifferentiated masses of cells with somatic proembryoids and embryoidsborne on suspensor structures proliferates from the scutellum of theseimmature embryos. The embryogenic callus isolated from the primaryexplant can be cultured on N6 medium and sub-cultured on this mediumevery 2 to 3 weeks.

The plasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst Ag,Frankfurt, Germany) may be used in transformation experiments in orderto provide for a selectable marker. This plasmid contains the Pat gene(see European Patent Publication 0 242 236) which encodesphosphinothricin acetyl transferase (PAT). The enzyme PAT confersresistance to herbicidal glutamine synthetase inhibitors such asphosphinothricin. The pat gene in p35S/Ac is under the control of the35S promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature313:810-812) and the 3′ region of the nopaline synthase gene from theT-DNA of the Ti plasmid of Agrobacterium tumefaciens.

The particle bombardment method (Klein et al. (1987) Nature 327:70-73)may be used to transfer genes to the callus culture cells. According tothis method, gold particles (1 μm in diameter) are coated with DNA usingthe following technique. Ten μg of plasmid DNAs are added to 50 μL of asuspension of gold particles (60 mg per mL). Calcium chloride (50 μL ofa 2.5 M solution) and spermidine free base (20 μL of a 1.0 M solution)are added to the particles. The suspension is vortexed during theaddition of these solutions. After 10 minutes, the tubes are brieflycentrifuged (5 sec at 15,000 rpm) and the supernatant removed. Theparticles are resuspended in 200 μL of absolute ethanol, centrifugedagain and the supernatant removed. The ethanol rinse is performed againand the particles resuspended in a final volume of 30 μL of ethanol. Analiquot (5 μL) of the DNA-coated gold particles can be placed in thecenter of a Kapton™ flying disc (Bio-Rad Labs). The particles are thenaccelerated into the corn tissue with a Biolistic™ PDS-1000/He (Bio-RadInstruments, Hercules Calif.), using a helium pressure of 1000 psi, agap distance of 0.5 cm and a flying distance of 1.0 cm.

For bombardment, the embryogenic tissue is placed on filter paper overagarose-solidified N6 medium. The tissue is arranged as a thin lawn andcovered a circular area of about 5 cm in diameter. The petri dishcontaining the tissue can be placed in the chamber of the PDS-1000/Heapproximately 8 cm from the stopping screen. The air in the chamber isthen evacuated to a vacuum of 28 inches of Hg. The macrocarrier isaccelerated with a helium shock wave using a rupture membrane thatbursts when the He pressure in the shock tube reaches 1000 psi.

Seven days after bombardment the tissue can be transferred to N6 mediumthat contains bialophos (5 mg per liter) and lacks casein or proline.The tissue continues to grow slowly on this medium. After an additional2 weeks the tissue can be transferred to fresh N6 medium containingbialophos. After 6 weeks, areas of about 1 cm in diameter of activelygrowing callus can be identified on some of the plates containing thebialophos-supplemented medium. These calli may continue to grow whensub-cultured on the selective medium.

Plants can be regenerated from the transgenic callus by firsttransferring clusters of tissue to N6 medium supplemented with 0.2 mgper liter of 2,4-D. After two weeks the tissue can be transferred toregeneration medium (Fromm et al. (1990) Bio/Technology 8:833-839).

Example 6 Expression of Chimeric Genes in Dicot Cells

A seed-specific expression cassette composed of the promoter andtranscription terminator from the gene encoding the β subunit of theseed storage protein phaseolin from the bean Phaseolus vulgaris (Doyleet al. (1986) J. Biol. Chem. 261:9228-9238) can be used for expressionof the instant polypeptides in transformed soybean. The phaseolincassette includes about 500 nucleotides upstream (5′) from thetranslation initiation codon and about 1650 nucleotides downstream (3′)from the translation stop codon of phaseolin. Between the 5′ and 3′regions are the unique restriction endonuclease sites NcoI (whichincludes the ATG translation initiation codon), SmaI, KpnI and XbaI. Theentire cassette is flanked by HindIII sites.

The cDNA fragment of this gene may be generated by polymerase chainreaction (PCR) of the cDNA clone using appropriate oligonucleotideprimers. Cloning sites can be incorporated into the oligonucleotides toprovide proper orientation of the DNA fragment when inserted into theexpression vector. Amplification is then performed as described above,and the isolated fragment is inserted into a pUC18 vector carrying theseed expression cassette.

Soybean embryos may then be transformed with the expression vectorcomprising sequences encoding the instant polypeptides. To inducesomatic embryos, cotyledons, 3-5 mm in length dissected from surfacesterilized, immature seeds of the soybean cultivar A2872, can becultured in the light or dark at 26° C. on an appropriate agar mediumfor 6-10 weeks. Somatic embryos that produce secondary embryos are thenexcised and placed into a suitable liquid medium. After repeatedselection for clusters of somatic embryos that multiplied as early,globular staged embryos, the suspensions are maintained as describedbelow.

Soybean embryogenic suspension cultures can be maintained in 35 mLliquid media on a rotary shaker, 150 rpm, at 26° C. with florescentlights on a 16:8 hour day/night schedule. Cultures are subcultured everytwo weeks by inoculating approximately 35 mg of tissue into 35 mL ofliquid medium.

Soybean embryogenic suspension cultures may then be transformed by themethod of particle gun bombardment (Klein et al. (1987) Nature (London)327:70-73, U.S. Pat. No. 4,945,050). A DuPont Biolistic™ PDS1000/HEinstrument (helium retrofit) can be used for these transformations.

A selectable marker gene which can be used to facilitate soybeantransformation is a chimeric gene composed of the 35S promoter fromCauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812), thehygromycin phosphotransferase gene from plasmid pJR225 (from E. coli;Gritz et al. (1983) Gene 25:179-188) and the 3′ region of the nopalinesynthase gene from the T-DNA of the Ti plasmid of Agrobacteriumtumefaciens. The seed expression cassette comprising the phaseolin 5′region, the fragment encoding the instant polypeptides and the phaseolin3′ region can be isolated as a restriction fragment. This fragment canthen be inserted into a unique restriction site of the vector carryingthe marker g ene.

To 50 μL of a 60 mg/mL 1 μm gold particle suspension is added (inorder): 5 μL DNA (1 μg/μL), 20 μL spermidine (0.1 M), and 50 pt CaCl₂(2.5 M). The particle preparation is then agitated for three minutes,spun in a microfuge for 10 seconds and the supernatant removed. TheDNA-coated particles are then washed once in 400 μL 70% ethanol andresuspended in 40 μL of anhydrous ethanol. The DNA/particle suspensioncan be sonicated three times for one second each. Five μL of theDNA-coated gold particles are then loaded on each macro carrier disk.

Approximately 300-400 mg of a two-week-old suspension culture is placedin an empty 60×15 mm petri dish and the residual liquid removed from thetissue with a pipette. For each transformation experiment, approximately5-10 plates of tissue are normally bombarded. Membrane rupture pressureis set at 1100 psi and the chamber is evacuated to a vacuum of 28 inchesmercury. The tissue is placed approximately 3.5 inches away from theretaining screen and bombarded three times. Following bombardment, thetissue can be divided in half and placed back into liquid and culturedas described above.

Five to seven days post bombardment, the liquid media may be exchangedwith fresh media, and eleven to twelve days post bombardment with freshmedia containing 50 mg/mL hygromycin. This selective media can berefreshed weekly. Seven to eight weeks post bombardment, green,transformed tissue may be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line may be treated as anindependent transformation event. These suspensions can then besubcultured and maintained as clusters of immature embryos orregenerated into whole plants by maturation and germination ofindividual somatic embryos.

Example 7 Expression of Chimeric Genes in Microbial Cells

The cDNAs encoding the instant polypeptides can be inserted into the T7E. coli expression vector pBT430. This vector is a derivative of pET-3a(Rosenberg et al. (1987) Gene 56:125-135) which employs thebacteriophage T7 RNA polymerase/T7 promoter system. Plasmid pBT430 wasconstructed by first destroying the EcoRI and HindIII sites in pET-3a attheir original positions. An oligonucleotide adaptor containing EcoRIand Hind III sites was inserted at the BamHI site of pET-3a. Thiscreated pET-3 aM with additional unique cloning sites for insertion ofgenes into the expression vector. Then, the NdeI site at the position oftranslation initiation was converted to an NcoI site usingoligonucleotide-directed mutagenesis. The DNA sequence of pET-3 aM inthis region, 5′-CATATGG, was converted to 5′-CCCATGG in pBT430.

Plasmid DNA containing a cDNA may be appropriately digested to release anucleic acid fragment encoding the protein. This fragment may then bepurified on a 1% low melting agarose gel. Buffer and agarose contain 10μg/ml ethidium bromide for visualization of the DNA fragment. Thefragment can then be purified from the agarose gel by digestion withGELase™ (Epicentre Technologies, Madison, Wis.) according to themanufacturer's instructions, ethanol precipitated, dried and resuspendedin 20 μL of water. Appropriate oligonucleotide adapters may be ligatedto the fragment using T4 DNA ligase (New England Biolabs (NEB), Beverly,Mass.). The fragment containing the ligated adapters can be purifiedfrom the excess adapters using low melting agarose as described above.The vector pBT430 is digested, dephosphorylated with alkalinephosphatase (NEB) and deproteinized with phenol/chloroform as describedabove. The prepared vector pBT430 and fragment can then be ligated at16° C. for 15 hours followed by transformation into DH5 electrocompetentcells (GIBCO BRL). Transformants can be selected on agar platescontaining LB media and 100 μg/mL ampicillin. Transformants containingthe gene encoding the instant polypeptides are then screened for thecorrect orientation with respect to the T7 promoter by restrictionenzyme analysis.

For high level expression, a plasmid clone with the cDNA insert in thecorrect orientation relative to the T7 promoter can be transformed intoE. coli strain BL21(DE3) (Studier et al. (1986) J. Mol. Biol.189:113-130). Cultures are grown in LB medium containing ampicillin (100mg/L) at 25° C. At an optical density at 600 nm of approximately 1, IPTG(isopropylthio-β-galactoside, the inducer) can be added to a finalconcentration of 0.4 mM and incubation can be continued for 3 h at 25°.Cells are then harvested by centrifugation and re-suspended in 50 μL of50 mM Tris-HCl at pH 8.0 containing 0.1 mM DTT and 0.2 mM phenylmethylsulfonyl fluoride. A small amount of 1 mm glass beads can be addedand the mixture sonicated 3 times for about 5 seconds each time with amicroprobe sonicator. The mixture is centrifuged and the proteinconcentration of the supernatant determined. One μg of protein from thesoluble fraction of the culture can be separated by SDS-polyacrylamidegel electrophoresis. Gels can be observed for protein bands migrating atthe expected molecular weight.

1. An isolated polynucleotide comprising: (a) a nucleotide sequenceencoding a polypeptide, wherein the amino acid sequence of thepolypeptide and the amino acid sequence of SEQ ID NO:24 have at least95% sequence identity based on the Clustal alignment method, or (b) thecomplement of the nucleotide sequence of (a), wherein the complement andthe nucleotide sequence of (a) contain the same number of nucleotidesand are 100% complementary; wherein a plant transformed with andexpressing said isolated polynucleotide exhibits an increasedaccumulation of oils in tissues of the plant when compared to a plantthat is not transformed with said polynucleotide.
 2. The polynucleotideof claim 1, wherein the amino acid sequence of the polypeptide and theamino acid sequence of SEQ ID NO:24 have at least 96% identity based onthe Clustal alignment method.
 3. The polynucleotide of claim 1, whereinthe amino acid sequence of the polypeptide and the amino acid sequenceof SEQ ID NO:24 have at least 98% identity based on the Clustalalignment method.
 4. The polynucleotide of claim 1, wherein thepolypeptide comprises the amino acid sequence of SEQ ID NO:24.
 5. Thepolynucleotide of claim 1 wherein the nucleotide sequence comprises thenucleotide sequence of SEQ ID NO:23.
 6. A vector comprising thepolynucleotide of claim
 1. 7. A recombinant DNA construct comprising thepolynucleotide of claim 1 operably linked to a regulatory sequence.
 8. Amethod for transforming a cell, comprising transforming a cell with thepolynucleotide of claim
 1. 9. A cell comprising the recombinant DNAconstruct of claim
 7. 10. A method for producing a plant comprisingtransforming a plant cell with the polynucleotide of claim 1 andregenerating a plant from the transformed plant cell.
 11. A plantcomprising the recombinant DNA construct of claim
 7. 12. A seedcomprising the recombinant DNA construct of claim
 7. 13. A method forisolating a polypeptide encoded by the recombinant DNA construct ofclaim 7, the method comprising: (a) transforming a host cell with therecombinant DNA construct of claim 9; (b) growing the transformed hostcell of (a) under conditions that are suitable for expression of therecombinant DNA construct; and (c) isolating the polypeptide from thetransformed host cell of (b).